The present invention relates to a nonlinear or multiphoton optical microscope equipped for the performance of imaging with good lateral resolution. It can be applied with particular benefit, but non-limitatively, to the quantitative imaging of collagen or of lipid content.
Generally, image resolution in nonlinear optical microscopy is linked to the square or the cube of the exciting intensity distribution. The usual nonlinear optical microscopes use a Gaussian-type excitation profile which is the natural distribution obtained with a non-shaped laser beam (Gaussian intensity profile and flat phase at the input to the focusing objective). The resolution is then from 300-400 nm in the lateral direction and from 1-3 microns in the axial direction, under usual conditions of excitation and detection (numerical aperture close to 1, excitation wavelength close to 1 μm). This good axial resolution is equivalent to a shallow depth of field and does not allow imaging in one go of a thin tissue section the thickness of which is a few microns (typically 2-7 μm). The larger the imaged field, the more critical this becomes. It is often essential, in particular in histology, to image large areas of interest (typically on the scale of centimeters) by moving the specimen and reconstituting a mosaic of images thereof. The imaged area must then remain flat and horizontal at the scale of this axial resolution of a few microns for the section to be imaged correctly. In practice this is never the case as microscope slides are not flat on this scale, the sections are never perfectly laid out, and it is very difficult to align the slide parallel to the focal plane within a few microns over distances of the order of a centimeter. FIGS. 2a and 2b show multiphoton imaging of a thin tissue section (typically 5 μm thick) having defects of flatness or of horizontality. FIG. 2a is according to the prior art and relates to the use of a strongly focused Gaussian beam (numerical aperture>0.5): the lateral resolution is satisfactory, but the depth of field is less than the thickness of the section and, as a result, the imaged volume sometimes extends beyond the tissue. FIG. 2b is according to the prior art and relates to the use of a weakly focused Gaussian beam (numerical aperture>0.5): the depth of field is greater, which makes it possible to keep the imaged volume within the tissue, but the lateral resolution is severely degraded.
Moreover, publications are known in the literature teaching the use of Bessel beams for extending the depth of field while retaining the good lateral resolution of an excitation beam. Among these publications, the following are distinguished:    “Two-photon excitation fluorescence microscopy with a high depth of field using an axicon”; Pascal Dufour et al., Applied Optics/vol. 45, No. 36/20 Dec. 2006;    “Scanning two photon fluorescence microscopy with extended depth of field”; E. J. Botcherby et al., Optics Communications 268 (2006) 253-260;    “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination”; Thomas A Planchon et al., Nature Methods/vol. 8 No. 5/May 2011; and    “Two-photon microscopy with simultaneous standard and extended depth of field using a tunable acoustic gradient-index lens”; Nicolas Olivier et al., Optics Letters/vol. 34, No. 11/Jun. 1, 2009.